Method and Device for Protein Delivery into Cells

ABSTRACT

Methods for performing surface-mediated protein delivery into living cells, and fabricating protein-transfected cell cluster arrays are provided. The method comprises providing a protein-containing mixture; depositing said protein-containing mixture onto a surface at defined locations; affixing the protein-containing mixture to the surface as microspots; and plating cells onto the surface in sufficient density and under conditions for the proteins to be delivered into the cells. The protein-containing mixture comprises any suitable amino acid sequence, including peptides, proteins, protein-domains, antibodies, or protein-nucleic acid conjugates, etc., with a carrier reagent. Protein-transfected cell arrays may be used for rapid and direct, screening of protein or enzymatic functions or any given intracellular protein interaction in the natural environment of a living cell, as well as for high-throughput screening of other biological and chemical analytes, which affect the functions of these proteins.

FIELD OF INVENTION

The present invention relates to biological arrays and assays. Moreparticularly, the invention pertains to methods and devices for usingsurface-mediated protein delivery to transfect living cells.

BACKGROUND

Genome sequencing projects have rapidly accelerated the pace of genediscovery and have led to the identification of thousands of new genesof higher order organisms, including humans. The challenge ahead is toidentify the biological functions of many of the newly discovered genes.DNA microarray—also known as “gene-chip”—technology has emerged as apowerful tool for genome-wide analysis of gene expression andgene-sequence variations. One caveat of microarray technology is thatprotein abundance within the cell does not always correlate withexpressed mRNA levels. Because the function of a gene is directlyrelated to the activity of its translated protein, an alternative andpossibly superior approach to elucidate gene functions lies in directanalysis of the functions of the specific proteins for which the geneencodes.

The current prevailing approach for analyzing protein function in vivois to employ cell-based assays. These types of assays are used to studythe function of one particular gene in a cellular context, through genetransfection and protein delivery. For the gene transfection approach,cells are transfected with a vector containing a specific gene thatleads to the overexpression of the gene product. With regard to theprotein delivery approach, cells are “transfected” with a functionalprotein, including antibodies, using membrane-disrupting, pore-formingreagents or other reagents, such as liposomes, as a carrier to deliverthe protein across the cell membrane. Using a variety of functionalassays, the effects of introduced DNA or proteins on cellular physiologyare then detected.

Protein delivery, i.e., protein transduction is the process by which apeptide or protein motif crosses the cell plasma membrane.Traditionally, methods to introduce antibodies, peptides or othermembrane-impermeable molecules into cells include micro-injection andelectroporation. The obvious disadvantages of these techniques are thatthey tend to be toxic to the recipient cells, they are non-specific(i.e., anything can enter or exit the cell once the membrane isdisrupted), and they exhibit low transfection efficiency and substantialvariability. To overcome the disadvantage associated with thesetechniques, researchers have developed a number of protein-transductiondomains (PTDs) that mediate protein delivery into cells. These PTDs orsignal peptide sequences are naturally occurring polypeptides of 15 to30 amino acids, which normally mediate protein secretion in the cells.They are composed of a positively charged amino terminus, a centralhydrophobic core and a carboxyl-terminal cleavage site recognized by asignal peptidase. Recently, researchers have shown that a number ofmembrane-translocating peptides can successfully mediate delivery ofpolypeptides, protein domains, and full-length protein, includingantibodies into cells using solution-based protein transfectionprotocols. Recently, researchers have also demonstrated the use of lipidliposomes or the like for protein delivery.

Traditionally, however, these approaches have been limited since theyare solution-based formats. Only one gene or protein may be studied perassay. As more there are more than 35,000 genes present in the humangenome, for instance, and approximately 10,000 of these genes areexpressed as proteins in any given cell type, a high-throughput methodfor studying gene function is needed.

SUMMARY OF THE INVENTION

The present invention describes a strategy, which involves the creationof protein-transfection cell arrays or microarrays, for thehigh-throughput analysis of protein functions in prokaryotic andeukaryotic cells. Protein transfection cell arrays may be used for rapidand direct screening of protein or enzymatic functions or any givenintracellular protein interaction in the natural environment of a livingcell. Moreover, the protein transfection cell arrays also are useful forhigh-throughput screening of other biological and chemical analytes,such as drugs, which affect the functions of these proteins. The proteinmay include an intracellular protein, cell-surface protein, biologicallyactive peptide, antibody, protein-nucleic acid conjugate,peptide-nucleic acid conjugate, fusion protein, synthetic peptide,protein-nanoparticle conjugate, protein-polymer conjugate, conjugatebetween a protein-organic chemical entity or protein-inorganic chemicalentity, multi-protein complexes, or any amino-acid containing moiety.

The invention also discloses a method for transfecting living cells withproteins using surface-mediated delivery. According to an embodiment ofthe method, a substrate surface having a protein of interest or aprotein to be introduced into cells, is used for culturing cells. Theprotein of interest or protein to be introduced into cells ispre-complexed with a carrier reagent before being applied to thesurface. Cells are then overlaid onto the prepared surface. The carrierreagent promotes the delivery of the protein of interest into the cell,thus transfecting the cells. Alternatively, proteins of interest areattached on a suitable substrate surface, then a carrier reagent isadded to the proteins to form complexes on the surface. In anotherembodiment, a fusion protein is used directly. The fusion proteincontains a protein or a protein domain of interest, fused covalentlywith any kind of protein or peptide that exhibits properties forspontaneous intracellular penetration (e.g., a herpes simplex protein,VP22). Preferably, a mixture containing a protein of interest and acarrier reagent includes a helper reagent to enhance the proteindelivery efficiencies. The present method produces a greater than 90%efficiency under optimized conditions for cell uptake of proteins. Thepresent surface-mediated protein delivery technique is also referred toas a “reverse protein delivery.”

The present invention also provides a method for fabricating an array oftransfected cell clusters having a set of proteins of interest. Theprotein-transfection cell-cluster array can be used for functionalscreening or phenotype screening. One embodiment of the method comprisesseveral steps. Provide a substrate with a surface. Provide a mixturesolution containing a protein and a carrier reagent. Immerse the tip ofa pin or other transfer device into the solution. Remove the tip of thepin from the solution with some of the solution adhering to the tip.Contact the substrate surface with the solution to transfer the solutionfrom the pin tip to the surface. Repeat the contacting step a pluralityof times to provide an array of protein microspots patterned on thesurface. Deposit or plate cells, which are in an appropriate medium suchas serum medium or serum-free medium, on top of the driedprotein-containing microspots to allow reverse delivery to occur. In avariation of the method using a solution of a protein, the methodfurther comprises incubating the protein microspots with a solutioncontaining a carrier reagent, then plating cells on top of the driedprotein-containing spots. The method can work also, mutatis mutandis,with a solution of a fusion protein that comprises a protein or aprotein domain of interest and a carrier reagent (e.g.,membrane-transducing peptides). A helper reagent may be also included inthe complex formed by the protein and the carrier reagent, to enhancethe protein delivery efficiencies. The helper reagent may be included inthe solution containing the carrier reagent or more preferably, thehelper reagent is pre-complexed with a protein before arrayed onto asurface. This method is referred to as a “living protein chip” or“living protein array” technology.

In an alternate embodiment, a protein array can be produced directlyfrom DNA templates arrayed on a surface using cell-free proteinsynthesis. The arrayed DNAs would contain the coding regions of desiredgenes in addition to the regulatory regions required fortranscription/translation and a tag moiety. Following conversion of theDNA array into protein using coupled transcription and translation, theproduced proteins would be immobilized on the surface by means of thetag in an ordered array format. The proteins of the resulting proteinarray would be delivered into cells using a carrier or translocationpeptide. The ability to generate arrays of cell clusters transfectedwith proteins synthesized directly from their corresponding DNAtemplates without having to purify the individual proteins a priorirepresents a significant advantage.

In another aspect, the present invention includes a cell array producedaccording to the present methods. A protein array having living cellscomprises the use of a pre-patterned surface that contains non-celladherent and cell-adherent areas. The protein mixture containing aprotein of interest and a carrier reagent is deposited onto thecell-adherent area. Other surfaces, such as microcolumn or micropillarstructures, may be also used for reverse protein delivery as describedin U.S. patent application Ser. Nos. 09/962,054 and 10/155,098,incorporated herein by reference.

Other features and advantages of the present method and array devicewill become evident from the following detailed description. It isunderstood that both the foregoing general description and the followingdetailed description and examples are merely representative of theinvention, and are intended to provide an overview for understanding theinvention as claimed.

BRIEF DESCRIPTION OF THE FIGURES

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawing(s) are available from thePatent and Trademark Office upon request.

FIG. 1 depicts a schematic rendering of the present method to deliverproteins into living cells by a “reverse deliver” technique. A mixturecontaining: 1) a protein-of-interest, 2) protein-of-interest complexedwith a carrier reagent, 3) a protein-of-interest-conjugate with acarrier reagent, or 4) a combination thereof, is deposited onto asurface (e.g., a glass slide, microcolumn, or the bottoms of wells in amulti-well plate) and dried to affix the proteins onto the surface.Cells are then desposited over the protein-containing spots, wherein thesurface bearing the protein is used to culture the cells. Underappropriate conditions, the proteins enter and transfect the cells.

FIG. 2 depicts a schematic rendering of the present method to deliverproteins into living cells to form a transfected cell cluster array. Aset of mixtures, containing a set of proteins of interest like in FIG.1, were arrayed onto a surface in defined, discrete or distinctlocations, and affixed to the surface. Cells plated and cultured overthe surface bearing the proteins are transfected when the proteins aredelivered into the cells, resulting in a transfected cell cluster array.A cell cluster array can be used in a variety of assays, such as forfunctional or phenotype screening.

FIGS. 3A and B are light microscopic images of CHO cells cultured on topof a dried, protein-containing spot on a GAPS-coated glass surface afterbeing fixed and stained. The protein mixture containedbeta-galactosidase (β-gal), a carrier (GP 41 peptide (HIV gp41 fragment519-541)), and a helper reagent (DEAE-dextran). Cells that have beenstained blue (darker) with x-gal solution are ones into which theproteins on the surface have successfully transfected. The blue colorindicates that the β-gal protein on the surface entered the cells and isstill fully functional. The dot line in the image of FIG. 3B, on theright, delineates the edge of the spot.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a surface-mediated, “reverse delivery”method of introducing proteins into living cells, in contrast toconventional techniques, wherein a cell culture is first providedfollowed by addition of a transfection solution containing proteins ofinterest to the cells. The present method affords researchers greatercontrol over the exact sequence, content, amount and nature of theprotein molecule. Not subject to the modifications and vagaries ofrelatively slow, in vivo protein synthesis, a researcher can pre-select,modify or check for uniformity of proteins prior to their introductioninto cells. By varying the amount of protein on the surface, researcherscan control the final intracellular protein concentration.

Recently, researchers have developed a microarray technique forexpressing multiple genes in an array format using “reversetransfection.” (Sabatini, D. & Ziauddin, J., “Microarrays of cellsexpressing defined cDNA,” Nature 411, 107-110 (2001); InternationalPatent Publication No. WO 01/20015, incorporated herein by reference.)According to their technique, a number of vectors, each containingspecific sets of genes mixed with a carrier such as gelatin, are arrayedonto a surface, treated with transfection reagents, and overlaid withmammalian cells. After a relatively long period of time (>24 hrs.), thecells that attach to the surface become transfected. The cellsoverexpress the genes corresponding to the sample cDNA, producing anarray of proteins in living cells, which can be used in gene functionalstudies. Unlike their technique, which is limited to gene encodingproteins, the present reverse protein delivery method can be used todeliver a broad range of proteins or protein-like biological molecules,including for example, functional peptides, antibodies, enzymes,particle-protein conjugates, and protein-complexes directly into a cell.

The present invention provides several advantageous and unique aspects,which differentiate it from other processes, including the reversetransfection method. A surface-mediated protein delivery to living cellsis able to transfect multiple proteins in a single assay using the arrayformat over traditional, solution-based cell protein deliverytechniques. This virtue provides for simultaneous, parallel analysis ofmany different proteins for a desired cellular readout (e.g., apoptosis,changes in cell morphology, effects on signaling pathway, etc.). Such ahigh-throughput capability signifies that many more proteins can bescreened per assay. The ability to screen more proteins per assay alsoreduces the amount of reagents consumed per assay, which can greatlyreduce assay costs.

Delivery of proteins into cells has certain advantage over deliveringDNA into cells by transfection. Since the protein molecule itself isbeing delivering into the cell and not the precursor gene, the presenttechnique bypasses the transcription-translation process associated withgene expression. Hence, the protein will begin performing its biologicalfunctions immediately after entry into the cell, greatly shortening theduration of time until cells can be assayed for protein function. Theshorter time period (<24 hrs.) required to manifest the effects of thedelivered protein in the cell is due in part to an ability to bypass thetranscription and translation process associated with gene expression.This feature is another advantage of the present method. Typically, onecan see changes within 12 hours. In some cases, greater than 95% ofproteins can be delivered in as little as 3-6 hours, or even as short aswithin 1 or 2 hours.

Another advantage of the method is that it can be used to assess therole of post-translational modification (PTM) on protein functions.Previously in DNA transfection methods, a protein would be modifiedafter translation according to specific signals on the protein (e.g.,glycosylation sites), which would be dependent on the availability of acorrect set of enzymes in the target cell line. The present methodcircumvents this need to find a cell line that will perform these PTMs.Using an array of the present device, one can test the effect thatdifferent PTMs in a protein (e.g., various sugar groups), which havebeen engineered in vitro, have on the biological function of the proteinin a cell, without need to mutate the DNA sequence, or alter the signalsand/or transfect the DNA into an appropriate cell line.

Moreover, unlike gene transfection that is limited to only theexpression of gene products, the present protein delivery approach canbe used to transfect cells with a much broader range of biologicals, forinstance, including bioactive peptides, proteins domains, proteins,antibodies, protein-nucleic acid conjugates, antibody-nucleic acidconjugates, nanoparticle-protein conjugates, multi-protein complexes,and any amino-acid containing moiety.

Furthermore, the method can make amenable studies of the same protein indifferent cells (e.g., a protein in differentiated and undifferentiatedstem cells). For instance, even though certain mammalian cells arenotoriously difficult to transfect, of the mammalian cell types testedto date according to the present invention, all were receptive toprotein transduction (delivery). Another advantage of the present methodis the ability to better control the biological effect by varying thedosage or quantity of protein per cell.

The invention is not limited to the particular embodiments of theinvention described below, as variations of the particular embodimentsmay be made and still fall within the scope of the appended claims. Itis also to be understood that the terminology employed is for thepurpose of describing particular embodiments, and is not intended to belimiting. Instead, the scope of the present invention will beestablished by the appended claims.

In the present specification and the appended claims, the singular forms“a,” “an,” and “the” include plural references unless the contextclearly dictates otherwise. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood to one of ordinary skill in the art to which this inventionbelongs.

Reverse Protein Delivery

As FIG. 1 depicts in schematic drawings, the method comprises in itssimplest form: depositing a protein-containing mixture onto a surface,and plating cells onto the surface in sufficient density and underappropriate conditions for delivering proteins in the protein-containingmixture into the cells. To illustrate, for example, mammalian cells arecultured on the mixture-coated surface. The cells take-up theprotein(s), which may affect cellular functions or observableexpression. A solution or mixture containing one or more kinds ofproteins of interest may be deposited and affixed to the surface, andused for subsequent cell culture and transfection.

The protein-containing mixture comprises known or unknown protein(s)(e.g., cell extracts) and a carrier reagent. A protein of interest, or aprotein to be introduced, may be a biologically active peptide, aprotein domain, an intracellular protein, an enzyme, a cell-surfaceprotein, a toxin, an antibody, an antibody-nucleic acid conjugate, aprotein-nucleic acid conjugate, a peptide-nucleic acid conjugate, afusion protein, a protein-nanoparticle conjugate, and a polymer. Thesurface bearing the protein-containing mixture can be used for culturingcells and, thereby, transfecting the adherent cells. The substrate usedin the present invention can comprise various glass, silicon, metal orpolymeric materials. The substrate can be configured as a bead, chip,slide, microwell plate, or a microcolumn device as described inco-assigned U.S. Provisional Application No. 60/317,660.

In an embodiment, the protein-containing mixture comprises protein and acarrier reagent that is present in an appropriate solvent, such asphosphate saline buffer. The protein is pre-complexed with the carrierreagent through physical interaction, for example, hydrophobicinteraction or electrostatic interaction, etc. The mixture is spottedonto a surface, thus producing a surface bearing (having affixedthereto) the protein-containing mixture in a defined location. Theresulting product is allowed to dry sufficiently, such that the spottedprotein-containing mixture is affixed to the surface and the spotsremain in the locations to which they become affixed, under theconditions used for carrying out subsequent steps in the method.Alternatively, the protein-containing mixture may further comprise ahelper reagent. The helper reagent is included to increase the proteindelivery efficiencies.

In another embodiment, a solution of a fusion protein is employeddirectly. The fusion protein comprises a conjugate of 2 parts: a proteinor a protein domain of interest and a carrier sequence or reagent. Thefusion protein solution is spotted onto a surface, thus producing asurface bearing (having affixed thereto) the fusion protein in a definedlocation. Again, the resulting product is allowed to dry sufficiently,such that the spotted protein-containing mixture is affixed to thesurface and the spots remain in the locations to which they becomeaffixed, under the conditions of use for subsequent steps in the method.Cells grown on the coated surface take up the protein, creatingtransfected adherent cells on top of the coated surface.

Alternatively, a mixture of fusion proteins (i.e., a protein or aprotein domain and a carrier reagent) is complexed with a helperreagent. The mixture is spotted onto a surface, to produce a surfacebearing (having affixed thereto) the fusion protein-containing mixturein a defined location. In a variation of the method, a solution of aprotein is spotted onto a surface and allowed to dry sufficiently. Asolution of a carrier reagent in the presence and absence of a helperreagent is applied to the protein-bearing surface to allow the proteinon the surface to interact more easily with the reagents, thus promotingprotein delivery into adherent cells occur.

In yet another embodiment of the method, a cell-free protein synthesisreaction can be performed, and the array of proteins produced is reactedwith a vehicle reagent to transport the proteins into cells. Proteinsare synthesized in situ on the coated surface of a substrate. Peptidesor proteins immobilized on the surface are preferably produced using invitro transcription/translation of DNA templates of particular genes,previously deposited on the surface. Since protein synthesis andpurification is both cumbersome and time-consuming, especially for thelarge number of proteins warranted for array applications, the presentmethod provides a simpler, faster method of producing and introducingproteins into cells. The DNA templates (representing full-length geneclones or gene fragments) can be produced either by PCR or RT-PCR usingoligonucleotide primers. The templates would include a tag moiety, sothat following conversion of the DNA into protein, using coupledtranscription and translation, the tagged proteins would becomeimmobilized on the surface. An example is a His-tag adhered to aNi-NTA-coated surface. These proteins would be delivered into cellsusing a carrier or translocation peptide. Enzymatic-aided cleavage at aprotein or protein cleavage site in the tag region, for instance, may beused to mediate the release of the synthesized proteins from the surfaceand entry into cells. Typical reagents used to perform this operationinclude glutathione-S-Transferase, thrombin and inteins. The inteinshave the advantage of being autocatalytic; that is, another enzyme isnot need to cleave off the protein of interest.

The production of the template DNAs can either be done prior to arrayingusing conventional PCR or RT-PCR, or the entire array of template DNAscan be synthesized on the substrate using solid-phase PCR byimmobilizing one set of primers on the substrate surface. (See,Andreadis, J. D. and Chrisey, L. A., Nuc. Acids Research 28, 2, e5(2000); He, M. and Taussig, M. J., Nuc. Acids Research 29, 15, e73(2001); International Patent Application No. WO 02/14860 A1). Thepresent cell-free, direct method of in situ protein synthesis obviatesthe need to produce and purify the hundreds or thousands of proteins ofinterest desired to transfect into cells. It also enables thetransfection into cells of domains or parts of proteins using specificPCR primers designed to produce the desired product.

Protein Arrays with Living Cells

As illustrated in FIG. 2, in an extension of the method, a solution ormixture containing a set of proteins of interest may be arrayed onto asurface in defined, distinct locations to create a rectilinearmicroarray or cluster array of cells with defined areas of adheredcells. Such microarrays may be employed in proteomic studies, such asfunctional or phenotype screening, or other high-throughput uses.

Depending on the desired use, living prokaryotic or eukaryotic cells(e.g., bacterial, mammalian, human, insect, or plant cells) may beplated in sufficient density onto the surface bearing theprotein-containing mixture. The protein samples are pre-spotted indefined and discrete locations. Under appropriate conditions, theprotein can be introduced into the cells. Preferably, the cells in anappropriate medium (either a serum or serum-free medium) are plated athigh density on top of the dried protein-containing spots, so as toincrease the likelihood that reverse delivery will occur. The proteinpresented in the protein-containing mixture affixed to the surfaceenters the cells. The resulting array of living cell clusters may beused to identify proteins that alter (enhance or inhibit) a pathway,such as a signaling pathway in a cell, or another property of a cell,such as its morphology.

As illustrated in FIG. 2, the array of the present invention includes asubstrate having a surface with a plurality of protein-containing probemicrospots. The microspots are affixed to the surface of a substrate. Asused herein, the term “affixed” means that the microspots maintain theirposition relative to the substrate under both cell culture andtransfection conditions. The means—covalent or electrostatic—by which aprotein of interest adheres is not necessarily limiting of theinvention. Cells that adhere to the top of each spot only uptake theproteins in the matrix arrayed on the surface to form a localized,transfected cell cluster array. Each probe microspot on the array maycomprise a protein of either known or unknown composition. The proteinpreferably is pre-complexed with a carrier reagent in either thepresence or absence of a helper reagent. Alternatively, the protein maybe first arrayed onto a surface, then interacted with a carrier reagentor a fusion protein comprising a protein or a protein domain and acarrier reagent, in the presence or absence of a helper reagent.

The probe microspots on the array may have any convenient shape, buttypically will be either circular, elliptoidal, oval, annular, or someother analogously rounded shape. The shape may, in certain embodiments,be a result of the particular method employed to produce the array,which is a non-limiting feature. The density of the microspots on thesurface of the substrate (i.e., both probe spots and non-probe spots;e.g., calibration spots, control spots, etc.) generally does not exceedabout 2000/cm², but is at least 1/cm² or 10/cm² to about 60/cm². Inparticular embodiments, the density does not exceed about 500/cm², andin certain preferred embodiments, the density does not exceed about400/cm² or about 300/cm². The microspots may be arranged generally inany convenient pattern over the surface of the solid or porous support.Typically, the pattern of spots will be present in the form of a grid,with rows and columns, across the surface of the substrate. Themicrospots, however, also may be arranged in a scatter or circularpattern, or the like.

In an embodiment of the array, each of the microspots of the array has adifferent protein. For instance, an array comprising about 100microspots could comprise about 100 different proteins. Likewise, anarray of about 10,000 microspots could comprise about 10,000 differentproteins. The protein(s) included on one microspot differs from theprotein(s) included on a second microspot of the same array. In such anembodiment, a plurality of different proteins is present on separatemicrospots of the array. An array may comprise at least about twodifferent proteins, but more typically about 10 different proteins. Morepreferably, the array comprises about 50 to 100 different proteins. Mostpreferably, arrays may comprise about 1,000-15,000 or more differentproteins.

In an alternative embodiment, each different protein is included on morethan one separate microspot on the array. For instance, each differentprotein optionally may be present on two to six different microspots. Anarray of the invention, therefore, may comprise about 3,000 microspots,but only comprise 1,000 different proteins since each different proteinis present on three different microspots. In a further alternativeembodiment, each of the microspots of the array comprises the sameprotein of interest but with different point mutations. The resultingarrays can be used to systematically examine the structure and functionrelationship of the protein in cells.

Additionally, in yet another alternative embodiment, the array comprisessubstantially identical microspots (i.e., microspots comprising the sameproteins) or a series of substantially identical microspots, which inuse are treated with a chemical or biological moiety (e.g., drug or drugcandidate). For example, an array of the invention can include a “miniarray” of 10-20 or more microspots, each microspot containing adifferent protein, wherein the mini array is repeated 20 times as partof the larger array.

The protein of one microspot may be different from that of another,however, the proteins may be related. One microspot may comprisemultiple, different proteins. For example, two different proteinsinvolved in the same or similar signaling pathways can be included inone microspot. In a preferred embodiment, the two different proteinsbelong to a same signaling pathway. The different proteins on theinvention array may be either functionally related or just suspected ofbeing functionally related. Such designs can be used for thematicmicroarrays.

When the function of the proteins are unknown, the different proteins onthe different microspots of the array may actually share a similarity instructure or sequence, or may be suspected of sharing a similarity instructure or sequence. Alternatively, the proteins may be fragments ofdifferent members of a protein family, or the proteins may sharesimilarity in pharmacological and physiological distribution or roles.

Proteins

A variety of conventional means may be applied to produce the proteinsused for reverse protein delivery in this invention. For instance, theprotein may be obtained from natural sources or, optionally, beoverexpressed using recombinant DNA methods. The protein may be eitherpurified using conventional techniques or left unpurified. A largenumber of proteins are available commerically, and may be used in thepresent invention.

As mentioned previously, proteins may include intracellular proteins,cell surface proteins, toxin proteins, antibodies, synthetic peptides,bioactive peptides, and protein domains; also protein-nucleic acidconjugates, and protein-nanoparticle conjugates, or multi-proteincomplexes. Any other biologicals including nucleic acids and polymersare also included in this invention. Additionally, conjugates between aprotein-organic chemical entity or protein-inorganic chemical entity(e.g., Biotin, fluorescent dies, silanol or silane derivatives, massspectrometry tags, or low-molecular weight chemical moiety, etc.) areincluded.

Examples of intracellular proteins include, but are not limited to:oxidoreductases, transferases, hydrolases, lyases, isomerases, ligases,kinases, phosphoproteines, and mutator transposons, DNA or RNAassociated proteins (for example, homeobox, HMG, PAX, histones, DNArepair, p53, RecA, robosomal proteins, etc.), electron transportproteins (for example, flavodoxins); adaptor proteins; initiatorcaspases, effector caspases, inflammatory caspases, cyclins,cyclin-dependent kinases, cytokeletal proteins, G-protein regulators,small G proteins, mitochondria-associated proteins, PDZ adaptorproteins, PI-4-kinases, etc. Recombinant proteins of unknown functionsmay also be used.

Applicable cell surface proteins include, but are not limited to:G-protein coupled receptors (e.g. the aderenergic receptor, angiotensinreceptor, cholecystokinin receptor, muscarinic acetylcholine receptor,neurotensin receptor, galanin receptor, dopamine receptor, opioidreceptor, erotonin receptor, somatostatin receptor, etc), G proteins,ion-channels (nicotinic acetylcholine receptor, sodium and potassiumchannels, etc), receptor tyrosine kinases (e.g. epidermal growth factor(EGF) receptor), immune receptors, integrins, and other membrane-boundproteins. Mutants or modifications of such proteins or proteinfunctional domains or any recombinant forms of such proteins may also beused.

Toxin proteins include, but are not limited to, cholera toxin, tetanustoxin, shiga toxin, heat-labile toxin, botulinum toxin A & E, deltatoxin, pertussis toxin, etc. Toxin domains or subunits may also be used.

Antibodies include, but are not limited to, organism-specific antibodiessuch as mouse and human antibodies, monoclonal and polyclonalantibodies, intact antibodies or single-chain antibodies.

Synthetic and bioactive peptides and protein domains also can be used totransfect cell using the method of the present invention. For example, asynthetic peptide comprising a sequence of AAYANAAVE may be used totransfect cells and monitor the protein tyrosine kinase (PTK) activityin cells. This peptide has recently been used as a universal PTKsubstrate for rapid detection of PTK activity in recombinant yeast(Clark, D. D. & Peterson, B. R., J. Am. Chem. Soc., 1, 207-209 (2002)).

Foreign proteins such as streptavidin and lectins and polymers may alsobe used.

Nanoparticle-protein conjugates may be used to transfect and visualizecells. The particles can include fluorescent tags, quantum dots, goldnanoparticles, paramagnetic nanoparticles, silica nanoparticles, orbeads of silica glass or polymer material, or the like. In otherembodiments, protein-DNA conjugates are used. The DNA is transcribed andtranslated into the resulting protein.

According to still another embodiment, the proteins may be synthesizedin situ on the surface of the substrate. Since protein synthesis andpurification of multiple proteins is both cumbersome and time-consuming,the present method provides a simpler, faster technique of producing andintroducing proteins into cells. Coupled cell-freetranscription/translation of DNA templates previously deposited on thesurface produces the corresponding proteins immobilized on the surface.One can deposit an array of DNA templates on a substrate surface,perform in situ protein synthesis on the surface and then treat theresulting protein array with a vehicle reagent to transport the proteinsinto cells.

By spotting a library of fusion proteins on an appropriate substratesurface, and then plating adherent cell lines on the surface it ispossible to assess the function of the protein in vivo in eukaryoticcells. Interactions between the delivered protein and other nativecellular proteins may be studied in vivo. Fusing the delivered proteinwith an auto-fluorescent marker, such as the green fluorescence protein(GFP), may monitor intercellular localization of the delivered protein.Reverse delivery into cells could be accomplished, for instance, usingVP22 fusion protein, herpes simplex protein, or any other protein withthe similar properties. The fusion protein can be made usingstate-of-the-art methods. Some commercially available vectors for theproduction of VP22 fusion protein in mammalian cells and Escherichiacoli (for example, pVP22/myc-His-2 from Invitrogen, Carlsbad, Calif.)are applicable. The fusion protein can be deposited directly and affixedonto the substrate surface, or the fusion protein can be pre-mixed witha helper reagent and then deposited and affixed onto the surface fortransfecting cells.

Further, fusion-proteins are preferably deposited and affixed onto anumber of distinct and defined locations, such as the bottom surface ofwells in a microplate. In another embodiment, a library of fusionproteins to be introduced into cells are preferably deposited andaffixed onto a number of defined and discrete locations of a surface totransfect cells. In one embodiment, the surface is a pre-patternedsurface that contains non-cell adherent and cell-adherent areas. Thefusion protein is deposited onto the cell-adherent area. In anotherembodiment, the fusion protein is deposited onto a number of defined anddiscrete locations in a surface, followed by treating the surface with abiological or a chemical to block these fusion protein-free surfaceareas from cell attachment.

Carrier Reagents

The particular embodiments of the invention are described in terms of acarrier reagent. Carrier reagents may comprise a variety of species. Inone embodiment, the carrier reagent is a bioactive cellmembrane-permeable reagent, or other peptides containingprotein-transduction domains (PTDs) (i.e., single peptide sequencescomprising about 15 to about 30 residues). Protein-transduction domains(PTDs) mediate protein secretion, and are composed of a positivelycharged amino terminus, a central hydrophobic core and acarboxyl-terminal cleavage site recognized by a single peptidase.Examples of such membrane-transducing peptides include Trojan peptides,human immuodeficiency virus (HIV)-1 transcriptional activator (TAT)protein or its functional domain peptides, and other peptides containingprotein-transduction domains (PTDs) derived from translocation proteinssuch as Drosophilia homeotic transcription factor Antennapedia (Antp)and herpes simplex virus DNA-binding protein, VP22, and the like. Somecommerically available peptides, for example, penetratin 1, Pep-1(Chariot reagent, Active Motif Inc., CA) and HIV GP41 fragment(519-541), can be used.

Other carrier reagents include signal sequences, which have been usedefficiently to target proteins to specific locations in both prokaryoticand eukaryotic cells, and a number of membrane-translocating peptides.Membrane-translocating peptides have been applied successfully tomediate membrane-translocation and the importation of a polpeptide,protein domain, full-length protein, or antibody into a cell usingstandard solution-based transfection protocols. The carrier reagent is abioactive peptide or ligand that can specifically bind to and activatecell surface receptors. After binding to the cell surface receptors, thereceptor and bound carrier-protein complex will undergo internalization,delivering ligand-protein complexes into cells. The proteins may becomplexed with the ligand beforehand or in situ. The ligand can becomplexed with the protein of interest or the protein to be introducedinto cells by means of non-covalent interaction such as hydrophobicinteraction or electrostatic interaction or both, or coupled covalentlyto the protein, or by means of a ligand-receptor binding interaction.For example, a carrier reagent can be modified with a ligand that canbind specifically to the protein of interest. To illustrate, a syntheticligand termed “Streptaphage” has efficiently delivered streptavidin tomammalian cells by promoting non-covalent interactions with cholesteroland sphingolipid-rich lipid raft subdomains of cell plasma membranes(Hussey, S. L. & Peterson, B. R., J. Am. Chem. Soc., 124, 6265-6273(2002)).

In another embodiment, the carrier reagent is a lipid liposome or thelike that can complex with a protein of interest and promote thedelivery of the protein into the cell. For example, the proteinencapsulated in the formulation binds to the negatively vehicle fordelivery (O. Zelphati et al., J. Bio. Chem., 276, 35103-19 (2001)).Products available commercially can be used, such as BioPORTER (GeneTherapy Systems), or ProVectin (Imgenex, San Diego, Calif.).

Protein delivery reagents (e.g., Chariot™ by Active Motif, or BioPORTER®by Gene Therapy Systems) can help save time by bypassing the traditionalDNA transfection, transcription and protein translation processesassociated with gene expression. Depending on the nature of theparticular reagent employed, fusion proteins or chemical coupling insome embodiments would not be needed. The reagent forms a complex withthe protein, stabilizes the macromolecule and protects it fromdegradation during delivery. Once internalized in a cell, the complexcan dissociate, leaving the macromolecule biologically active and freeto proceed to its target organelle.

Helper Reagents

The particular embodiments of the invention are described in terms of ahelper reagent. In one embodiment, the helper reagent is a polymer suchas DEAE-dextran, dextran, polylysine, and polyethylamine. In anotherembodiment, a helper reagent can also be a cell adherent-enhancingprotein, such as fibronectin and gelatin. The helper reagent can be asugar-based gelatin (e.g., polyethylene glycol) or a synthetic orchemical-based gelatin, such as acrylamide. In a further embodiment, thehelper reagent can be a RGD peptide, such as Arg-Gly-Asp-Ser,Arg-Gly-Asp-Ser-Pro-Ala-Ser-Ser-Lys-Pro, and the like. Alternatively,the helper reagent can be a mixture of a hydrogel and a RGD peptide, andcombination of any the aforementioned molecules. The use of helperreagents enhances the efficiency of protein delivery into the cells.

Substrates

The substrates used in the present invention for arrays comprise atleast one surface on which a pattern of probe spots (protein-containingmixture microspots) may be affixed. The surface may be either solid orporous. Also, the surface may be either smooth and substantially planar,or have irregularities, such as depressions or elevations. The surfaceon which the pattern of spots is present can be modified with one ormore different layers of compounds, which serve to change thesurface-chemistry properties in a desirable fashion. For example, thesurface may be coated with chemical molecules that can enhance thebinding of the protein-containing mixture, while simultaneously stillallowing the proteins to be transfected into cells, which are overlaidon top of the protein-containing mixture microspots. For instance, acoating of γ-aminopyropyl silane (GAPS) or polylysine may be applied toa glass surface.

The substrate may be fabricated from a ceramic, glass, metal, plastic,polymer or co-polymer, crystalline, or conductive material (e.g., indiumtin oxide), or any combinations thereof; alternatively, the surface mayhave a coating of one of such materials on another. Such substratesinclude, but are not limited to, for example (semi) noble metals such asgold or silver; glass materials such as soda-lime glass, pyrex glass,vycor glass, quartz glass; metallic or non-metallic oxides; silicon,monoammonium phosphate, and other such crystalline materials; transitionmetals; plastics or polymers, including dendritic polymers, such aspoly(vinyl chloride), poly(vinyl alcohol), poly(methyl methacrylate),poly(vinyl acetate-maleic anhydride),poly(dimethylsiloxane)monomethacrylate, polystyrenes, polypropylene,polyethyleneimine; copolymers such as poly(vinyl acetate-co-maleicanhydride), poly(styrene-co-maleic anhydride), poly(ethylene-co-acrylicacid) or derivatives of these, or the like.

The substrate may take a variety of configurations depending on theintended use of the array, ranging from simple to complex. Formicroarray uses, the substrate can be a slide or plate, such as of arectangular or disc configuration. Alternatively, a standard, multiwellmicroplate can have spots deposited in each well. In many embodiments,the substrate will have a rectangular shape in cross-section with alength of from about 20-200 mm, and a width of from about 10-150 mm.

Particular embodiments of the surface include the bottom of a well in asingle or multi-well microplate or strip-of-wells of any commerciallyavailable format (e.g., 6, 8, 12, 24, 48, 96, 192, 384, 576, etc.).Aside from a microplate, the substrate surface may be that of amicrocolumn or micropillar device, such as described in U.S. ProvisionalApplication No. 60/317,660, the content of which is incorporated hereinin its entirety. Microcolumns can have a variety of shapes, such as acube, cylinder, cone, frustoconical or polygonal body.

Preparation of the Arrays

Arrays for the present invention may be prepared using a multiplicity ofmicro-patterning techniques. In one embodiment, the tip of a mechanicalprojection or probe (also referred to as a “pin”) is immersed into asolution of a protein-containing mixture. The tip is removed from thesolution with an amount of solution adhered to the tip. The wetted tipmakes contact with the surface of a substrate, thereby transferring thesolution from the tip to the surface.

A “pin” as applied according to the present invention may be of anyshape, size, and dimension. For example, the pin printing process mayinvolve ring shaped pins, square pins, or point pins, etc. In anotherembodiment, the direct contact printing may involve single pin-printingor multiple pin-printing, that is, a single pin printing methodinvolving a source plate or multiple pin-printing using a laid out arrayof multiple pins patterned in any format.

The printing apparatus may include a print head, plate, substratehandling unit, XY or XYZ positioning stage, environmental control,instrument control software, sample tracking software, etc. Such anapparatus may include, for example, a quill pin-printer available fromCartesian Technologies, Inc.

For a high-density array, a typographical probe array having a matrix ofprobes may be used to align and fit each probe from the matrix into acorresponding source well (e.g., a well of a microtiter plate).

A variety of other techniques may also be used to produce the array ofprotein-containing mixtures of the present invention. For example,arrays of the present invention can be produced using microstamping(U.S. Pat. No. 5,731,152), microcontact printing using PDMS stamps(Hovis 2000), capillary dispensing devices (U.S. Pat. No. 5,807,522) andmicropipetting devices (U.S. Pat. No. 5,601,980). For radioactive assaysusing arrays of protein-containing mixtures, pipette-based liquidtransfer techniques are preferred for fabricating the arrays becausesuch techniques can give rise to spots of larger size with a range ofseveral hundred microns to several millimeters.

Use of the Array

Once a substrate is prepared, bearing an array comprising a plurality ofmicrospots of protein-containing mixtures, cells are plated or otherwiseplaced onto the substrate surface in sufficient density and underappropriate conditions for the introduction of the proteins into thecells. Clusters of live cells that have taken up a protein at eachlocation, i.e., transfected with the proteins, will cover the array. Asmentioned before, the present invention can be applied to a variety ofcells, including eukaryotic cells, such as mammalian cells (e.g., human,monkey, mouse, etc.), bacterial, insect or plant cells. Preferably, thecells (in a serum or serum-free medium) are plated on top of the driedprotein-containing spots at high density (e.g., 0.5-1×10⁵/cm²), in orderto increase the likelihood that reverse transfection occurs.Alternatively, the density of cells may vary from about 0.3×10⁴/cm² toabout 5×10⁵/cm²; or, in other embodiments, from about 2×10⁵/cm² to about3×10⁵/cm²; or, from about 0.5×10⁴/cm² to about 2×10⁵/cm².

Transfected cell microarrays can be of broad utility for high-throughputstudy of biological function of proteins, as well as screening compoundsthat affect the specific function of the proteins in a cell.Particularly, transfected cell arrays are useful for probing singletransduction pathways, blocking transcription factors, for conductingdetailed structure-function analysis of integrin and other receptors'cytoplasmic domains, and for drug discovery. A variety of techniques maybe employed to detect the effects of the protein of interest onrecipient cells (i.e., cells that have been delivered of the protein).These techniques may include, for instance, immunofluorescence, in whicha fluorescently-labeled antibody that binds a protein of interest isused to determine if the protein is present in cells.

Moreover, a microplate-based reverse protein delivery technique can beemployed for cell culture and localized cell transfection. A microplatemay be pre-coated with protein-containing mixtures, or a certain amountof a solution containing a protein of interest pre-complexed with acarrier reagent can be deposited onto the bottoms of each single wells,allowing the mixture dry and affixed on the surface of each well.

Another application of the present method targets the use of“protein-transfection” to express proteins in primary cell lines, whichare difficult to transfect using classical DNA-based techniques.According to this embodiment, DNA-constructs expressing the protein ofinterest are first transfected or otherwise introduced into a populationof relatively “easy to transfect” cells, such as, but not limited toHeLa, 3T3, HEK 293, COS. Afterwards, this first population of cells isdesposited onto a surface. Using an immobilized cell has an advantage ofamplifying in-vivo protein production before deliver into primary cells.After either in situ lysis of these cells and local capture of theseproteins, such as using a tag, or in situ excretion of the expressedprotein of interest onto the surface, primary cells can be added and aretransfected by the fusion proteins in a local manner. Then, a secondpopulation of primary cells is deposited over the proteins present onthe surface, and the proteins are delivered into the primary cells.Alternatively, the method can work with arrays of fusion proteins, asdescribed above, are delivered into relatively “easy to transfect”cells. The result is a surface or an array of primary cells, eachexpressing a different fusion protein.

The pertinent content of each of the articles and publications madereference to in the present specification is incorporated herein byreference.

EXAMPLE

The following empirical example further describes and illustrates thepresent invention. The particular example, described in terms ofproteins, is not limiting of the present invention. It is understoodthat any suitable amino acid sequence, including peptides, proteins,antibodies, or protein-nucleic acid conjugates, is encompassed by thepresent invention. The proteins will have an effect on a cellularfunction or interact with a cellular component.

Example Reverse Delivery of β-Galactosidase into Cells Materials

The materials used included: β-galactosidase (grade VIII, purified fromE. Coli), RGD peptides gly-arg-gly-asp-ser, andgly-arg-gly-asp-ser-pro-lys), dextran (M.W. 45000 Da), and DEAE-detran(M.W. 40000 Da) (Sigma Chemical (St. Louis, Mo.)); Chariot (Pep-1)(Active Motif Inc, Carlsbad Calif.); and HIV GP-41 fragment (519-541)(Bachem (King of Prussia, Pa.)); and Gamma-Amino Propyl Silane (GAPS)slides were obtained from Coming Inc (catalog #2550) (Corning, N.Y.).Cell culture media was obtained from Gibco. A β-galactosidase stainingkit was obtained from Qiagen. Other chemicals were from Sigma.

Method Stock Solution Preparations

In 10 mM PBS buffer (pH 7.4), β-galactosidase was dissolved to aconcentration of 0.25 mg/ml, and stored at 4° C. Translocation peptides(i.e., carrier peptide), Chariot or GP41 fragment, were dissolved in 60%DMSO to a concentration of 2 mg/ml, and stored at −20° C. before use.Dextran or DEAE-dextran was dissolved into ddH₂O to give a concentrationof 2%, and stored at 4° C. RGD peptides was dissolved in PBS buffer togive a concentration of 1 mg/ml, and stored at −20° C. before use.Dextran, DEAE-dextran and RGD peptides were used as a helper reagent toenhance the delivery efficiencies.

Diluting and Mixing

The β-galactosidase stocking solution was diluted into PBS buffer togive final protein concentration of 0.1 mg/ml. Peptide stock solutionwas diluted into PBS buffer to give a final concentration of 0.4 mg/ml,and subject to sonication to avoid self-aggregation. Two dilutedsolutions were gently mixed together using equal volume. Helper reagentcan be also added into the mixture (optimized concentrations for thesehelper reagent are 1 mg/ml for dextran and DEAE-dextran, 0.1 mg/ml forRGD peptides). The resulted mixture was incubated at room temperaturefor about 30 minutes to one hour.

Spotting

On a slide surface coated with γ-amino-propylsilane (Corning Inc.,Corning, N.Y.), 20 spots in four separated grids, each having 5replicated spots within a single grid, was made using 5 μl of themixture solution, and dried at room temperature for one hour.

Storage

The slides bearing protein-containing spots, in some cases, were storedat 4° C. in nitrogen for several days to several weeks, with noobservable loss of protein transfection efficiencies or functionality ofthe proteins inside the transfected cells.

Cell Type and Culture Conditions

Human Embryonic Kidney (HEK) 293T cells and CHO cells cultured inIscove's Modified Dulbecco's Medium (IMDM) supplemented with 10% fetalbovine serum.

Cell Culture and Transfection

In a tissue culture hood, a suspension containing about 0.5-2×10⁵ cells(CHO or HEK) was plated or otherwise deposited on top of each gridcontaining separated protein spots. The cell suspension either containedserum or was serum-free. For transfection, the cells attached and grewfor 3 hours under 5% CO₂ and 95% humidity.

Fixation of Cells

After culturing, the slides were twice rinsed very gently with PBSbuffer, and then fixed in 4% formaldehyde/PBS solution for 3 minutes,and again rinsed twice with PBS buffer.

X-Gal Staining

Following the standard protocol recommended by the supplier, reagentsolution from a β-galactosidase staining kit (Invitrogen) were added tothe fixed cells, and incubated with the cells for 30 minutes at 37° C.

Cell Examination

Using a light microscope, the percentage of cells stained blue color wascounted to calculate the protein transfection efficiencies withβ-galactosidase. The stained cells could be stored after disposing ofthe staining solution and overlaying with 70% glycerol.

As illustrated in FIG. 3, certain percentages of the cells on the top ofthe protein-containing spots turned blue after X-gal staining,suggesting that these proteins on the surface entered the cells, andthese proteins are still fully functional inside the cells. As evidencedby the appearance of blue color after X-gal staining, the cells of bothcell lines become transfected when deposited on top of the mixture spotscontaining beta-gal and delivery vehicle (VP41 fragment). The reversedelivery process took a relatively short time. Reaction typically takesplace within 3 hours. Protein delivery into the cells appears to behighly efficient. Cells plated on the protein-mixture spots for as shorta time as only 3 hours become transfected. Although not required to useof serum-free medium, the use of serum-free medium does improve thetransfection efficiency of the reverse protein delivery process. Thepresence of DEAE-dextran, dextran, RGD peptide(Gly-Arg-Gly-Asp-Ser-Pro-Lys) or the combination of these reagents alsoimproves the efficiency. In some experiments the cells attached to themixture spots were trypsinized following X-gal staining and replated ina microplate well. Significantly, the blue x-gal color remained withinthe cells, strongly suggesting that that the blue color is indeedintracellular, derived from the fully-functional β-gal delivered intothe cells.

The present invention has been described in detail by way of examples.Persons skilled in the art, however, may appreciate that modificationsand variations may be made to the present method and device withoutdeparting from the scope of the invention, as defined by the appendedclaims and their equivalents.

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 27. A reverse delivery method ofintroducing proteins into living cells comprising: spotting a protein ofinterest on a surface in defined and discrete locations; drying andaffixing the protein spot; treating the protein spot with a carrierreagent; depositing cells onto the surface; and transporting saidprotein into said cells.
 28. The method according to claim 27, whereinsaid protein in the spot interacts and complexes with said carrierreagent.
 29. The method according to claim 27, wherein said protein ofinterest includes a bioactive peptide, protein-domain, intracellularprotein, enzyme, cell surface protein, toxin protein, antibody,antibody-nucleic acid conjugate, protein-nucleic acid conjugate,peptide-nucleic acid conjugate, protein-nanoparticle conjugate, orpolymer.
 30. The method according to claim 27, wherein said protein ofinterest includes a fusion protein that contains a translocation domainand a protein domain of interest.
 31. The method according to claim 27,wherein the carrier reagent is a naturally occurring or synthetictranslocation peptide, or a liposome.
 32. The method according to claim27, wherein said carrier reagent includes: a bioactive cellmembrane-permeable reagent, a membrane-translocating peptide, a peptidecontaining protein-transduction domains (PTDs), a signal sequence, abioactive peptide, or a ligand that can bind specifically to andactivate cell surface receptors.
 33. The method according to claim 27,wherein said carrier reagent includes: Trojan peptides, humanimmuodeficiency virus (HIV-1) transcriptional activator (TAT) protein orits functional domain peptides, Drosophilia antennapedia homeotictranscription factor, and herpes simplex virus-1 DNA binding proteinVP22.
 34. The method according to claim 27, wherein saidprotein-containing mixture further comprises a helper reagent.
 35. Themethod according to claim 34, wherein said helper reagent includes: apolymer, a cell adherent-enhancing protein, a sugar-based gelatin, asynthetic or chemical-based gelatin, a RGD peptide, a mixture of ahydrogel and a RGD peptide, or a combination of any the aforementionedmolecules.
 36. The method according to claim 34, wherein said helperreagent is a polymer selected from the group consisting of:DEAE-dextran, dextran, polylysine, and polyethylamine.
 37. The methodaccording to claim 34, wherein said helper reagent is a celladherent-enhancing protein selected from the group consisting of: afibronectin and gelatin.
 38. The method according to claim 34, whereinsaid helper reagent is either polyethylene glycol or acrylamide.
 39. Themethod according to claim 34, wherein said helper reagent is a RGDpeptide selected from the group consisting of: Arg-Gly-Asp-Ser (SEQ IDNO: 2), Arg-Gly-Asp-Ser-Pro-Ala-Ser-Ser-Lys-Pro (SEQ ID NO: 3), and thelike.
 40. The method according to claim 27, wherein said conditions fordelivery is in a serum-free medium.
 41. The method according to claim27, wherein said conditions for delivery is in a serum-containingmedium.
 42. The method according to claim 27, wherein said surface isthat of a microtiter plate well-bottom, a microcolumn, a slide, a strip,a bead, a particle, or nanoparticle.
 43. The method according to claim27, wherein said defined locations comprise an array of microspots onsaid substrate surface.
 44. The method according to claim 43, whereinsaid defined locations each contains a different protein.
 45. The methodaccording to claim 43, wherein each different protein is included onmore than one separate microspot on the array.
 46. The method accordingto claim 43, wherein said microspots on the array comprises the sameprotein of interest but with different point mutations.
 47. The methodaccording to claim 45, wherein the different proteins are functionallyrelated.
 48. The method according to claim 43, wherein said array form athematic microarray.
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 60. A high-throughputbiological analysis device fabricated according to the method of claim49.
 61. An array produced according to a method comprising: providing aprotein-containing mixture; depositing said protein-containing mixtureonto a surface at defined locations; affixing said protein-containingmixture to the surface; and plating said cells onto the surface.
 62. Thearray according to claim 61, wherein said protein containing mixture isa solution of: a) protein-of interest; b) a protein-of-interestcomplexed with a carrier reagent; c) a conjugate of a protein-ofinterest and a carrier reagent; or a mixture of (b) and (c).
 63. Thearray according to claim 62, wherein said protein-of-interest includes abioactive peptide, protein-domain, intracellular protein, enzyme, cellsurface protein, toxin protein, antibody.
 64. The array according toclaim 62, wherein said protein-of-interest includes an antibody-nucleicacid conjugate, protein-nucleic acid conjugate, peptide-nucleic acidconjugate, protein-nanoparticle conjugate, protein-polymer conjugate,conjugate between a protein-organic chemical entity or protein-inorganicchemical entity, multi-protein complexes, or any amino-acid containingmoiety.
 65. The array according to claim 62, wherein said conjugate is aprotein-of-interest covalently attached to a carrier reagent.
 66. Thearray according to claim 62, wherein said protein-of-interest includes afusion protein that contains a translocation domain and a protein domainof interest.
 67. The array according to claim 62, wherein the carrierreagent is a naturally occurring or synthetic translocation peptide. 68.The array according to claim 66, wherein said carrier reagent includes:a bioactive cell membrane-permeable reagent, a membrane-translocatingpeptide, a peptide containing protein-transduction domains (PTDs), asignal sequence, a bioactive peptide, or a targeted ligand that can bindspecifically to and activate cell surface receptors.
 69. The arrayaccording to claim 67, wherein said carrier reagent includes: Trojanpeptides, human immuodeficiency virus (HIV-1) transcriptional activator(TAT) protein or its functional domain peptides, Drosophiliaantennapedia homeotic transcription factor, and herpes simplex virus-1DNA binding protein VP22.
 70. The array according to claim 61, whereinsaid protein-containing mixture further comprises a helper reagent. 71.The array according to claim 69, wherein said helper reagent includes: apolymer, a cell adherent-enhancing protein, a sugar-based gelatin, asynthetic or chemical-based gelatin, a RGD peptide, a mixture of ahydrogel and a RGD peptide, or a combination of any the aforementionedmolecules.
 72. The array according to claim 69, wherein said helperreagent is a polymer selected from the group consisting of:DEAE-dextran, dextran, polylysine, and polyethylamine.
 73. The arrayaccording to claim 69, wherein said helper reagent is a celladherent-enhancing protein selected from the group consisting of: afibronectin and gelatin.
 74. The array according to claim 69, whereinsaid helper reagent is either polyethylene glycol or acrylamide.
 75. Thearray according to claim 69, wherein said helper reagent is a RGDpeptide selected from the group consisting of: Arg-Gly-Asp-Ser (SEQ IDNO: 2), Arg-Gly-Asp-Ser-Pro-Ala-Ser-Ser-Lys-Pro (SEQ ID NO: 3), and thelike.
 76. The array according to claim 61, wherein said surface is thatof a microtiter plate well-bottom, a microcolumn, a slide, a strip. 77.The array according to claim 61, wherein said surface is made of aceramic, glass, metal, polymer or co-polymer, crystalline, or conductivematerial, or any combinations thereof.
 78. The array according to claim76, wherein said surface may have a coating of any one of said materialson another.
 79. The array according to claim 61, wherein said definedlocations comprise an array of microspots on said substrate surface. 80.The array according to claim 61, wherein said defined locations eachcontains a different protein.
 81. The array according to claim 79,wherein each different protein is included on more than one separatemicrospot on the array.
 82. The array according to claim 80, whereinsaid microspots on the array comprises the same protein of interest butwith different point mutations.
 83. The array according to claim 79,wherein the different proteins are functionally related.
 84. The arrayaccording to claim 61, wherein said array is a thematic microarray. 85.A transfected-cell cluster array made according to a surface-mediatedprotein delivery method, the method comprising: providing DNA templateson a substrate surface; synthesizing, in situ on said surface, proteinsfrom said DNA templates; affixing said proteins to said surface;reacting said synthesized proteins with a carrier reagent; adding cellsto said surface; and transporting said proteins into said cells.
 86. Thearray according to claim 84, wherein said protein synthesizing step isperformed as a cell-free reaction.
 87. The array according to claim 84,wherein said protein synthesizing step uses coupled transcription andtranslation processes directly from corresponding DNA templates.
 88. Thearray according to claim 84, wherein said DNA templates include a tagmoiety.
 89. The array according to claim 84, wherein said DNA templatesrepresent full-length gene clones or gene fragments.
 90. The arrayaccording to claim 84, wherein said DNA templates is produced either byPCR or RT-PCR using oligonucleotide primers.
 91. The array according toclaim 84, wherein said method further comprises cleaving at a proteincleavage site in a region of said tag moiety to release said synthesizedproteins from said surface for entry into said cells.
 92. The arrayaccording to claim 84, wherein said array is a thematic array.
 93. Thearray according to claim 84, wherein the carrier reagent is a naturallyoccurring or synthetic translocation peptide.
 94. The array according toclaim 84, wherein said carrier reagent includes: a bioactive cellmembrane-permeable reagent, a membrane-translocating peptide, a peptidecontaining protein-transduction domains (PTDs), a signal sequence, abioactive peptide, or a targeted ligand that can bind specifically toand activate cell surface receptors.
 95. The array according to claim84, wherein said carrier reagent includes: Trojan peptides, humanimmuodeficiency virus (HIV-1) transcriptional activator (TAT) protein orits functional domain peptides, Drosophilia antennapedia homeotictranscription factor, and herpes simplex virus-1 DNA binding proteinVP22.
 96. The array according to claim 84, wherein saidprotein-containing mixture further comprises a helper reagent.
 97. Thearray according to claim 95, wherein said helper reagent includes: apolymer, a cell adherent-enhancing protein, a sugar-based gelatin, asynthetic or chemical-based gelatin, a RGD peptide, a mixture of ahydrogel and a RGD peptide, or a combination of any the aforementionedmolecules.
 98. The array according to claim 95, wherein said helperreagent is a polymer selected from the group consisting of:DEAE-dextran, dextran, polylysine, and polyethylamine.
 99. The arrayaccording to claim 95, wherein said helper reagent is a celladherent-enhancing protein selected from the group consisting of: afibronectin and gelatin.
 100. The array according to claim 95, whereinsaid helper reagent is either polyethylene glycol or acrylamide. 101.The array according to claim 95, wherein said helper reagent is a RGDpeptide selected from the group consisting of: Arg-Gly-Asp-Ser (SEQ IDNO: 2), Arg-Gly-Asp-Ser-Pro-Ala-Ser-Ser-Lys-Pro (SEQ ID NO: 3), and thelike.
 102. The array according to claim 84, wherein said surface is thatof a microtiter plate well-bottom, a microcolumn, a slide, a strip, abead, a particle, or nanoparticle.
 103. The array according to claim 84,wherein said surface is made of a ceramic, glass, metal, polymer orco-polymer, crystalline, or conductive material, or any combinationsthereof.
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